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Original Citation:
Effectiveness of aerobiological dispersal and microenvironmental requirements together influence spatial colonization patterns of lichen species on the stone cultural heritage
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DOI:10.1016/j.scitotenv.2019.06.238 Terms of use:
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Highlights
Relationship of lichen patterns on heritage stones and propagule dispersal is tested We combined analyses of the spatial population structure with aerobiological analyses Dispersal patterns and microenvironment co-drive specific abundance and distribution Knowledge on lichen reproductive patterns is crucial for stone heritage conservation *Highlights (for review : 3 to 5 bullet points (maximum 85 characters including spaces per bullet point)
Abstract 1
Dispersal patterns of lichen species in monumental and archaeological sites and their 2
relationships with spatial population structure are almost unknown, hampering predictions on 3
colonization dynamics that are fundamental for planning conservation strategies. 4
In this work, we tested if the local abundance and distribution pattern of some common lichen 5
species on carbonate stones of heritage sites may be related to their patterns of propagule 6
dispersal. We combined analyses of the spatial population structure of eight species on the 7
calcareous balustrade of a heritage site in Torino (NW Italy) with aerobiological analyses. In 8
situ and laboratory analyses were mainly focused on the ejection of ascospores and their air 9
take-off and potential dispersal at short and long distance. Results indicate that the spatial 10
distribution of lichens on the stone surfaces is influenced by both species-specific patterns of 11
propagule dispersal and microenvironmental requirements. In particular, apotheciate species 12
that have a higher ejection of ascospores with higher potential for long range dispersal are 13
candidate for a much aggressive spreading on the monumental surfaces. Moreover, their 14
occurrence on natural or artificial stone surfaces in the surroundings of the stone monumental 15
surface may easily support recolonization dynamics after cleaning interventions, as an 16
effective supply of propagules is expected. On the other hand, species with a lower dispersal 17
rate have a more clustered distribution and are less effective in rapid recolonization, thus 18
representing a minor threat for cultural heritage conservation. These results support the idea 19
that information on the reproductive strategy and dispersal patterns of lichens should be 20
coupled with traditional analyses on stone bioreceptivity and microclimatic conditions to plan 21
effective restoration interventions of stone surfaces. 22
Keywords 23
calcareous stone; cultural heritage conservation; lichen colonization dynamics; ascospore 24
ejection; reproductive strategy; spatial population structure 25
*Manuscript (double-spaced and continuously LINE and PAGE numbered)-for final publication Click here to view linked References
1. Introduction 27
Since the 1990s, scientists dealing with the conservation of stone cultural heritage focused 28
their attention on bioreceptivity, i.e. the ability of a material to be colonized by living 29
organisms (Guillitte, 1995; Miller et al., 2012). Stone bioreceptivity depends on intrinsic 30
properties of the lithology, including physical and chemical features (primary receptivity), and 31
may also be affected by the influence of early colonizers, starting colonization successions 32
(secondary receptivity), or by human interferences, as the application of restoration products 33
(tertiary receptivity) (Guillitte, 1995; Guillitte and Dreesen, 1995). However, lithobiontic 34
colonization also depends on extrinsic factors, as microclimate conditions and the supply of 35
reproductive structures, for which atmospheric aerosol is generally considered the main vector 36
(Caneva et al., 2003a; De Nuntiis and Palla, 2017). 37
Aerobiology applied to cultural heritage examines airborne biological particulates which may 38
be a potential cause of biodeterioration, when the characteristics of the substrate and the 39
surrounding (micro-)environmental conditions are compatible with the ecological and 40
nutritional needs of the lithobiontic microorganisms (Caneva et al., 2003a). In this context, 41
most of investigations dealt with indoor environments, where the characterization of 42
bioaerosols was combined with the control of microclimatic conditions (Skóra et al., 2015; De 43
Nuntiis and Palla, 2017). A minor attention has still been devoted to outdoor environments, 44
where knowledge on the load of biodeteriogens in the bioaerosol may possibly support a 45
better understanding of population dynamics and consequent threats to heritage conservation 46
(Caneva et al., 2003b; Piervittori et al., 2007). In particular, lichens, which are among the 47
most aggressive biodeteriogens of the outdoor stone cultural heritage (Seaward, 2015; 48
Salvadori and Casanova-Municchia, 2016), have been rarely considered in aerobiological 49
investigations (Favero-Longo et al., 2014; Banchi et al., 2018). In this perspective, the 50
possibility to characterize lichen ascospores in the air spora and to explore relationships 51
between specific dispersal ability and local distribution patterns on heritage surfaces has been 52
recently demonstrated (Morando et al., 2017). However, dispersal patterns of lichen species 53
most frequently found in monumental and archaeological sites, and their relationships with 54
spatial population structure, are almost unknown, hampering predictions on colonization 55
dynamics that are fundamental for planning conservation strategies. 56
In this work, we tested the null hypothesis that the local abundance and distribution pattern of 57
common lichen species on carbonate stones of heritage sites are independent from their 58
microclimate requirements and/or their patterns of propagule dispersal. We combined 59
analyses of the spatial population pattern of eight species on the calcareous balustrades of a 60
heritage site in Torino (NW-Italy) with aerobiological analyses. Establishment and growth of 61
lichens imply the success of the whole aerobiology pathway (Scheidegger and Werth, 2009), 62
including propagule discharge, take-off, transport (dispersal s.s.), deposition and impact 63
(Lacey, 1996; Magyar et al., 2016), and, in the case of asymbiotic propagation, the acquisition 64
of a compatible photobiont (Honegger, 2012). In situ and laboratory analyses were mainly 65
focused on the ejection of ascospores (and the detachment of vegetative propagules) and their 66
air take-off and potential dispersal at short and long distance. 67
2. Material and Methods 68
2.1 Study sites and lichen survey 69
Investigations were carried out on the balustrade of the podium of the Basilica of Superga 70
(WGS 84: N 45°4.826' - E 7°46.047'), symbol of Baroque architecture in NW Italy (Fig. 1a; 71
Corino et al. 1990), located on a top of the hill (672 m a.s.l.) on the east side of the Torino 72
metropolitan area. In this area, mean annual temperature is 12.5 °C and mean annual rainfall 73
is 900 mm. The balustrade (Fig. 1b), approx. 100 m long and 30 cm wide, borders the podium 74
of the Basilica and is made of a local biocalcirudite known as Gassino stone, the carbonate 75
sedimentary rock most widely used in Torino architecture (Borghi et al., 2014). Last cleaning 76
intervention on the balustrade dates back to 1990 (Corino et al., 1990). 77
Lichen colonization on the top horizontal surfaces (capstones) of the balustrade was recorded 78
in March 2016 by surveying rectangular plots (30 × 150 cm) distributed in the South (n=5), 79
South-West (n=4), North-West (n=4) and North sides (n=5; Fig. 1c-d). 80
All the plots shared lithology, horizontal orientation, surface micromorphology and 81
conservation history. The solar exposure of each plot, related to different levels of shading by 82
the Basilica, was estimated from hemispheric photographs, taken with a LG 360 CAM (R-83
105; LG Electronics inc., South Korea), processed with CAN-EYE software (v. 6.495; Weiss 84
and Baret, 2017) and evaluated with regard to the portion (%) of the sky non-covered by the 85
building (Supplementary note S1). Distance from the nearest trees (meters) was also 86
calculated, as increasing proximity could be related to increasing eutrophication of the 87
balustrade surface (Nascimbene et al. 2009a). 88
Each plot was surveyed using a grid divided into three lines of 15 quadrats (10 × 10 cm). 89
Small samples of lichen thalli were collected by non-destructive techniques to check in the 90
laboratory provisional identifications assigned during fieldwork. Lichen species were 91
identified using Clauzade and Roux (1985), Smith et al. (2009) and monographic 92
descriptions. Nomenclature follows Nimis (2016). Sample vouchers were deposited at the 93
Cryptogamic Herbarium of the University of Torino (HB-TO Cryptogamia). 94
95
2.2 Population pattern analyses 96
Population pattern analyses were carried out for the eight more abundant species: 97
Candelariella aurella (Hoffm.) Zahlbr., Circinaria calcarea (L.) A. Nordin, SaviÉ& Tibell, 98
Circinaria contorta (Hoffm.) A. Nordin, Savić & Tibell, Myriolecis albescens (Hoffm.) 99
Pyrenodesmia erodens (Tretiach, Pinna & Grube) Søchting, Arup & Frödén, Verrucaria 101
macrostoma DC., and Xanthocarpia crenulatella (Nyl.) Frödén, Arup & Søchting. 102
For each species, the following abundance (i-ii) and distribution (iii-iv) parameters were 103
considered: 104
(i) total frequency, as % occurrence in the 18 rectangular plots throughout the balustrade; 105
(ii) average frequency, as mean % occurrence in the 10 × 10 cm quadrats of each plot; 106
(iii) Moran’s I coefficient (Moran, 1950), to evaluate the species distribution pattern along the 107
balustrade (distance matrix in Table S1) according to a clustered or diffuse/random spatial 108
structure, and computed using the R-software Version 3.5.1 (The R Foundation for Statistical 109
Computing, 2018), ape package (Paradis et al., 2004); 110
(iv) an average Spread Index (SpIn), here proposed to evaluate whether the species have a 111
clustered or diffuse distribution at the scale of each plot, and computed for each plot as 112
follows: 113
SpIn = Avfull/Avempty 114
where Avfull is the average number of colonized quadrats surrounding each colonized quadrat, 115
and Avempty is the average number of colonized quadrats surrounding each uncolonized 116
quadrat (details on the SpIn calculation in Supplementary note S2). 117
Differences among species in terms of average frequency per plot and Spread Index were 118
tested by ANOVA with post-hoc Tukey’s test using SYSTAT 10 (P<0.05 as significant). 119
Potential relationships of population patterns with the microenvironmental parameters varying 120
along the balustrade were also considered. In particular, Pearson correlation of species 121
frequency with solar exposure (CorrSol) and distance from nearest trees (CorrTree) were 122
calculated, on the basis of data quantified for each plot, using SYSTAT 10 (post-hoc 123
Bonferroni’s test; P<0.05 as significant). Ecological indicator values proposed by Nimis 124
(2016) on the tolerance range of each species with regard to light and eutrophication were 125
considered for comparison. 126
127
2.3 Dispersal analysis 128
The aerobiology pathways of the species were examined in situ with regard to the release of 129
reproductive structures, and in the laboratory considering their air take-off and potential 130
dispersal at short and long distance. 131
The ejection of ascospores and, in the case of P. erodens, the release of soredia (i.e. vegetative 132
propagules) were evaluated directly on the balustrade by placing overturned Petri dishes 133
containing agar medium (15 g l-1Agar A1296, Sigma Aldrich Chemie, Steinheim, Germany, 134
in deionized water; Petri diameter = 5.5 cm; agar layer 6 mm high) on both areas colonized by 135
the different species and areas lacking colonization (control). Petri dishes were positioned 136
after that thalli had been hydrated for one hour with patches of sterile paper soaked with 137
deionized water. Agar surfaces were exposed on the balustrade surface for three hours, during 138
which air flow allowed the progressive desiccation of thalli. For each species, replicates 139
(n=15) were performed in autumn-winter 2016 and in summer 2018, placing the Petri dishes 140
in different parts of the balustrade. The number of apothecia covered by each Petri dish was 141
counted by image analysis of high quality photographs taken with a Nikon Coolpix AW100, 142
using ImageJ (1.51g; US National Institutes of Health, Bethesda, USA). The ascospores were 143
counted under a Nikon Eclipse 50i microscope (400× magnification). 144
Air take-off and the consequent short and long distance potential dispersal of reproductive 145
structures were evaluated following the protocol described by Favero-Longo et al. (2014) and 146
Morando et al. (2017). Apothecia of the epilithic species C. aurella, C. calcarea, C. contorta, 147
M. albescens, and X. crenulatella, and of the endolithic M. dispersa, were collected from the 148
balustrade and pasted on an adhesive tape at the bottom of Petri plates in groups of 100-400 149
units, to be used for the experiments. Fertile thalli of the epilithic peritheciate V. macrostoma 150
and the endolithic P. erodens were instead collected from natural outcrops due to difficulties 151
in sampling intact reproductive structures from the balustrade without affecting the stone 152
substrate. In the laboratory, for each species, apothecia (or whole thalli of V. macrostoma and 153
P. erodens) were immersed for 15 min in deionized water and incubated for three days within 154
a perspex box (120 x 70 x 60 cm) including a fan (FT30-G1, Viglietta SPA, Italy) moving the 155
air at about 4 m sec-1, 10 Petri dishes (diameter = 5.5 cm) containing agar medium and a 156
Hirst-type volumetric sampler VPPS 2010 (Lanzoni, Italy), typically used for aerobiological 157
monitoring. The Petri dishes, encircling the rocks at a distance of 5-15 cm from the exposed 158
reproductive structures, were used as passive traps to capture short-range dispersed 159
propagules. With the VPPS, located with the suction nozzle at 40 cm from the ground layer 160
and at 50-70 cm from the apothecia, the propagule dispersion in the air volume was examined, 161
the spore take-off and entering the turbulent air being considered a requisite for long-range 162
dispersal. For each species, the experiment was replicated two times, due to limitations in the 163
sampling of intact apothecia from the balustrade. The ascospores that impacted on the VPPS 164
adhesive tape and on the agar surfaces were quantified at 400x magnification according to 165
Favero-Longo et al. (2014). 166
Population structure parameters, dispersal parameters (maxima values of ejected spores, and 167
spores involved in short- and long distance dispersal) and microenvironment-related 168
parameters (CorrSol, CorrTree) calculated for each species were indexed and processed 169
through a Redundancy Analysis (RDA) to summarize the joint variations, choosing forward 170
selection of variables option (Legendre et al., 2011). Each parameter was indexed with 171
reference to an ordinal scale (from 1 to 10), with ranges defined dividing in ten parts the 172
interval between the maximum and minimum observed values. In the case of micro-173
environmental parameters, the absolute values were used for the indexing procedure, as both a 174
positive or a negative correlation indicated the influence of the variable conditions on the 175
population patterns. RDA was performed using CANOCO 4.5 (Ter Braak & Šmilauer, 2002). 176 177 3. Results 178 3.1 Population patterns 179
The balustrade of the podium of the Basilica was abundantly colonized by lichens (11 species 180
in total), with the 8 selected species occurring in at least 50% of the plots (Fig. 2a). With 181
reference to the ecological indicator values (Table 1), the requirement of all these species with 182
respect to the light factor ranges from plenty of diffuse light (light index=3) to a very high 183
direct solar radiation (5). With respect to their tolerance to eutrophication, Circinaria 184
calcarea and Pyrenodesmia erodens are only resistant to a weak eutrophication (max 185
eutrophication index=3), while other species occur in rather to highly eutrophicated 186
conditions (max eutrophication index=4-5). In substantial agreement, from a syntaxonomic 187
point of view all the species are diagnostic of the class Verrucarietea nigrescentis, including 188
calcicolous (hemi-)nitrophilous lichen associations, with the exception of P. erodens, which is 189
diagnostic species of Clauzadeetea immersae, including non or slightly nitrophilous 190
associations (Roux et al. 2009; syntaxonomic scheme in Table S2). 191
The population structure reflects approx. 25 years of recolonization dynamics following the 192
last cleaning intervention. Candelariella aurella and Myriolecis dispersa were the most 193
abundant in terms of both total frequency and average frequency per plot (100% of the plots 194
and >60% of quadrats per plot, respectively; Fig. 2b-c). With regard to this latter parameter, 195
the other species were in a range between 7% (Verrucaria macrostoma) and 31% 196
(Xanthocarpia crenulatella) and most of the slight differences were not statistically 197
significant. All the thalli exhibited a healthy appearance, with the exception of those of P. 198
erodens, most of which were ailing and partially covered by cyanobacteria. 199
All species occurred in at least one plot of each plot group, at the differently exposed sides of 200
the balustrade, with the exception of C. calcarea which was absent in the S-exposed corner. 201
Moran I’ coefficients for C. calcarea, X. crenulatella, M. albescens, M. dispersa, and P. 202
erodens indicated a clustered distribution pattern even if the values were low (<0.3), 203
suggesting that the spatial autocorrelation was not strong (Fig. 2d). At the plot level, values of 204
the Spread Index were significantly higher for C. aurella than for C. calcarea (0.8) and P. 205
erodens (0.5), indicating diffuse and clustered distribution respectively (Fig. 2e). The other 206
species had intermediate SpIn values in the range 0.9-1.1. 207
Correlation coefficients higher than ǀ0.5ǀ were calculated for frequencies per plot and solar 208
exposure (CorrSol) with regard to C. calcarea (-0.765), M. dispersa (-0.637) and M. 209
albescens (-0.560), and for distance of nearest trees (CorrTree) in the case of X. crenulatella 210
(-0.524; Table 1). However, only the negative CorrSol of C. calcarea, indicating its higher 211
frequency in more shaded plots, resulted significant (P<0.05). 212
3.2 Specific reproductive potential 213
Significant differences in the quantity of reproductive structures dispersed by the eight species 214
were found, with remarkable variability for both the ejection and the short- and long-distance 215
dispersal steps (Fig. 3). 216
In situ tests showed that all the six apotheciate species and the peritheciate V. macrostoma 217
ejected ascospores (Fig. 3a). The strong variability between different replicates seems to be a 218
prominent trait of some species. Most of the ascospores were still arranged in packages (in 219
most cases still including eight ascospores), while the ejection of single spores was a less 220
frequent phenomenon (Fig. 4a-c). C. aurella displayed the highest values of maximum, 75th 221
percentile and median ejection, accounting for an ejection capability significantly higher than 222
M. albescens, M. dispersa, and V. macrostoma (in the range 0.13-0.16 ejected spores per 224
apothecium, sp·ap-1) were 2.6 to 5.3 times higher than those of C. contorta (0.05 sp·ap-1) and 225
C. calcarea (0.03 sp·ap-1). High values of ascospore ejection, however, were also observed 226
for two replicates of this latter species (up to 0.7 sp·ap-1). No soredia, characterizing the 227
asexual reproduction of P. erodens, were captured or observed. 228
In laboratory assays, all the apotheciate species displayed potential for both short- and long-229
distance dispersal, while dispersed reproductive structures were not collected for the 230
peritheciate V. macrostoma and the sorediate P. erodens (Fig. 3b). For all the species, single 231
ascospores rather than ascospore packages, were collected by both the passive traps and the 232
volumetric sampler (Fig. 4d-g). Short distance dispersal was mostly recorded on Petri dishes 233
in lee position with respect to the exposed apothecia, indicating that ascospore split and 234
deposition immediately followed the package discharge (data not shown). Notwithstanding 235
that only two replicates for each species were performed, some differences among the 236
apotheciate species were worth noting. M. albescens showed the highest value of spores 237
involved in short-range dispersal (1.2 sp·ap-1), approx. the double of the maxima observed for 238
M. dispersa and X. crenulatella, and three and four times higher than those of C. aurella and 239
Circinaria sp. pl. respectively. With regard to spores available for long-range dispersal, M. 240
dispersa (1.3 sp·ap-1) and C. aurella (1.2 sp·ap-1) had the highest values, followed by X. 241
crenulatella (0.7 sp·ap-1) and, with approx. three times lower values, M. albescens and 242
Circinaria sp. pl. (0.4 sp·ap-1). 243
In the RDA (Fig. 5a-b), the first three axes explained 97.1% of the variance of relationship 244
between population structure, dispersal and microenvironment-related parameters. Total 245
frequency through the plots, average frequency per plot, Spread Index (for which higher 246
values indicate a higher species diffusion) were positively related along the first axis (66.3% 247
of total variance) with ejected spores, spores available for long-distance dispersal and 248
CorrTree. The Moran I’s coefficient was positively related with CorrSol and the spores 249
involved in short-distance dispersal along the second (25.7%) and third (5.1%) axes, 250
respectively. Spores available for long-distance dispersal and CorrSol displayed the highest 251
conditional factor (F=5.30 and F=4.33, respectively; P<0.05) according to forward selection, 252
followed by CorrTree (F=2.67), spores involved in short-distance dispersal (F=1.50) and 253
ejected spores (F=1.13), which were not significant (P>0.05). C. aurella and M. dispersa, 254
scattered in the right side of the diagrams (Fig. 5a-b), were positively associated with spores 255
available for long-distance dispersal. In the left upper side, characterized by the Moran I’s 256
coefficient, C. calcarea and M. albescens related with CorrSol and spores involved in short-257
distance dispersal, respectively. P. erodens, V. macrostoma, and C. contorta scattered in the 258
left lower side of the diagrams, oppositely to spores available for long-distance dispersal and 259
CorrSol. Similar relationships were observed in two other RDAs considering the median and 260
75thpercentile values of spore ejection rather than the maximum (data not shown). 261
262
4. Discussion 263
Our findings reject the tested null hypothesis and indicate that both microclimate 264
requirements and specific patterns of propagule dispersal contribute to determine the local 265
abundance and spatial patterns of epilithic and endolithic lichens encrusting calcareous 266
heritage surfaces. Since in our study site the last cleaning intervention dates back to 1990 267
(Corino et al., 1990), these results may provide a keystone reference for elucidating 268
recolonization dynamics following restoration activities. 269
In previous literature (Ariño et al., 1995; De los Ríos et al., 2009; McIlroy de la Rosa et al., 270
2013), spatial distribution of lichens on heritage structures was related to microenvironmental 271
conditions. This could apply also to our balustrade where species frequencies per plot were 272
partially related to different levels of solar exposure and distance from nearest trees, 273
responsible for eutrophication (Nascimbene et al. 2009a). The significant negative CorrSol of 274
C. calcarea, and the highest value of CorrTree of X. crenulatella, likely reflect different 275
microenvironmental requirements which may account for the spatial autocorrelation recorded 276
for these species. Higher frequency of X. crenulatella with increasing proximity to trees fits 277
the high nutrient requirement of this nitrophilous species (Nimis 2016). Instead, despite the 278
significant negative value of CorrSol for C. calcarea, ecological indicator values suggest for 279
this species a higher tolerance of direct light radiation than that of V. macrostoma and X. 280
crenulatella, which show positive CorrSol values. Both these incongruencies -for which it 281
should be taken into account that ecological indicator values are expert assessments (Nimis 282
2016)- and the weak statistical support to the calculated CorrSol and CorrTree (mostly with 283
P>0.05) reflect the fact that the eight species have comparable microenvironmental 284
requirements. Moreover, these evidences suggest that species spatial patterns may be also 285
related to other factors as in the case of the quantified differences in the reproductive potential 286
(Ellis, 2019). Actually, RDA results showed that both spores available for long-distance 287
dispersal, i.e. a reproduction-related factor, and CorrSol, i.e. a microenvironment-related 288
factor, were significant determinants of the population structure. In particular, the positive 289
correlation of species abundance with spores available for long-distance dispersal and ejected 290
spores underlines the need of a high release of reproductive structures to successfully 291
establish and spread colonization (Scheidegger and Werth, 2009). Species ejecting a higher 292
number of ascospores, which are also more easily taken off and dispersed, as C. aurella and 293
M. dispersa, are the most abundant on the balustrade and are therefore the main candidate as 294
deterioration agents. They have great potential for effective and rapid recolonization after 295
cleaning interventions if fertile thalli occur in the surroundings of the heritage site, even at 296
long distance. For other species, as M. albescens, the patchy and clustered distribution, is 297
related to a high number of spores involved in short-distance dispersal, but with a low 298
potential for long-distance dispersal. Such species, even if present in the nearby of restored 299
heritage surfaces, are expected to be less aggressive in terms of recolonization potential and 300
more easily controlled. 301
As ascospore packages were mostly detected in ejection tests, while single spores were 302
collected in dispersal assays, the air movement was likely responsible for a drying effect 303
determining the package disaggregation. It is worth noting that experiments without air 304
movement generally yielded the discharge at a short distance of spores still organized in 305
packages (Garrett, 1971; Ostrofsky and Denison, 1980). Maxima values of spores available 306
for long-distance dispersal were observed for the small spores of C. aurella (major axis 10-12 307
µm) and M. dispersa (major axis 8-14 µm), according to the fact that particles with lower 308
diameters always show lower deposition rate and higher life times in the air than larger ones 309
(De Nuntiis et al., 2003). Similarly, higher colonization rates and effective dispersal were 310
related to a lower propagule size for epiphytic lichens (Hilmo et al., 2012; Johansson et al., 311
2012) as well as for crustose aquatic lichens (Nascimbene et al., 2009b). However, ascospore 312
size may be not the only factor determining the amount of spores available for long-distance 313
dispersal, as values measured for the small spores of M. albescens (major axis 8-14 µm) are 314
lower than those for the large spores of Circinaria sp. pl. (major axis 20-30 µm). With this 315
regard, ascospores of similar size were shown to be dispersed at different distances in 316
laboratory experiments in absence of air movement (Bailey and Garrett, 1968). Nevertheless, 317
this phenomenon would require further clarification. 318
In the case of P. erodens, the release of soredia was related to its active dissolution of the 319
substratum and the consequent surface exposure to dispersal vectors (Tretiach et al., 2003). 320
However, dispersal of soredia is usually associated to dry surface conditions or to the impact 321
of rain drops (Armstrong, 1991), while the washing strategies we adopted in situ and in the 322
lab, stimulating ascospore discharge, may have instead prevented the discharge of propagules 323
of P. erodens. Because of their larger size (av. diameter approx. 50 µm) than ascospore (max 324
lentgh: 30 µm in C. calcarea) the soredia should be suitable for short distance dispersal, as 325
generally reported for asexual propagules (Scheidegger and Werth, 2009). This would account 326
for the clustered distribution of this species at both the whole balustrade and plot scale. In this 327
perspective, this species could be critical for heritage conservation due to its endolithic 328
growth (Pinna and Salvadori, 2000) and resiliance to restoration interventions (Sohrabi et al., 329
2017), rather than for its dispersal and recolonization potential. In the case of the Basilica of 330
Superga, in particular, the unhealthy condition of P. erodens suggests that current 331
environmental conditions are less favourable for the species than in the past and that its thalli 332
may be remnants dating back the last cleaning intervention, and not removed because of their 333
endolithic growth. 334
The low abundance of the peritheciate V. macrostoma on the balustrade is consistent with 335
poor successes in the ejection tests and the absence of spores in laboratory dispersal assays. 336
The absence of ascospores of V. macrostoma even in the passive traps may indicate a 337
limitation of the distance of discharge within the 5-15 cm from the nearest source, which is 338
compatible with the weak discharge capability quantified for some species (Bailey and Garrett 339
1968). Nevertheless, we cannot rule out that dispersal by V. macrostoma may depend on 340
vectors other than air, as animals, feeding of its thalli (which do not contain deterrent 341
secondary metabolites) and thus becoming responsible of zoochory (Baur et al. 1995; 342
Asplund 2010). The low abundance of V. macrostoma on the balustrade may also depend on a 343
low availability of compatible photobionts (Spitale and Nascimbene 2012): V. macrostoma 344
has photobiont partners (e.g. Coccobotrys, Protococcus; Stocker-Wörgötter and Turk 1989) 345
different from those of the genus Trebouxia that are shared by the other species (Smith et al. 346
2009). However, the fact that V. macrostoma is more abundant on other calcareous 347
balustrades of cultural heritage sites in Torino (Supplementary note S3), suggests that local 348
conditions may affect fertility and reproductive performance of the species (Monte, 1993). 349
5. Conclusive remarks 352
Results of our quantitative analyses indicate that the spatial distribution of lichens on the 353
balustrade of the Basilica of Superga -representative of calcareous heritage surfaces in the dry 354
submediterranean area of Italy (sensu Nimis 2016)- is correlated with species-specific patterns 355
of propagule dispersal, as revealed by aerobiological experiments, and microenvironmental 356
requirements. In particular, apotheciate species that have a higher ejection of ascospores with 357
higher potential for long range dispersal are candidate for a much aggressive spreading on the 358
monumental surfaces. Moreover, their occurrence on natural or artificial stone surfaces in the 359
surroundings of the stone monumental surface may easily support recolonization dynamics 360
after cleaning interventions, as an effective supply of propagules is expected (Favero-Longo 361
et al., 2014). When these species occur in the surroundings, a restoration plan more effective 362
in the long term should thus consider the adoption of protocols to decrease the bioreceptivity 363
of the monumental surface (Pinna, 2017; Fidanza and Caneva, 2019) and/or, if practicable 364
(e.g. in the case of a localized occurrence), their removal. On the other hand, species with a 365
lower dispersal rate have a more clustered distribution and are less effective in rapid 366
recolonization, thus representing a minor threat for cultural heritage conservation. When they 367
occur on surfaces adjacent to or in the close proximity of the monument (at a distance in the 368
order of few meters or less), their removal appears similarly recommendable to drastically 369
reduce the risk of recolonization. When they occur at greater distances, their presence may be 370
instead safely considered as a biodiversity value for the often invoked joined valorization of 371
natural and cultural heritage (Nimis et al., 1992). 372
All these results strongly support the idea that information on the reproductive strategy of 373
lichens, and, in particular, the evaluation of their local dispersal patterns should be coupled 374
with traditional analyses on stone bioreceptivity and microclimatic conditions to plan 375
effective restoration interventions of stone surfaces. 376
Acknowledgements 377
Field and laboratory activities were carried out thanks to funds from the ‘Università di 378
Torino’ and ‘Compagnia di San Paolo’ (Project CSTO169034/2016: ‘From rocks to stones, 379
from landforms to landscapes – geoDIVE’ - PROactive management of GEOlogical heritage 380
in the PIEMONTE Region). MM was a recipient of a PhD fellowship funded by INPS 381
(Istituto Nazionale di Previdenza Sociale). The authors are grateful to Comune di Torino, 382
Soprintendenza Archeologia Belle Arti e Paesaggio per la Città Metropolitana di Torino and 383
Direzione del Polo Museale del Piemonte for permission to access the monumental sites. The 384
authors are also thankful to Irene Franciosa, Chiara Michelis and Giulio Borghi (Università di 385
Torino) for assistance during fieldwork. The authors are grateful to the Editor E. Paoletti and 386
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Figure captions 550
Fig. 1. Study site: a the Basilica of Superga (Torino, NW Italy) viewed from SW; b the 551
balustrade of the podium of the Basilica; c distribution of the relevés (n=18, dots) along the 552
balustrade (scale bar = 5 m); d lichens on the top horizontal surface of the balustrade, covered 553
with the 10 × 10 cm square grid. 554
Fig. 2. Lichen abundance and distribution on the balustrade of the Basilica of Superga. a 555
Presence/absence of the surveyed species (Candelariella aurella, Can.aur; Myriolecis gr. 556
dispersa, Myr.dis; Myriolecis albescens, Myr.alb; Verrucaria macrostoma, Ver.mac; 557
Circinaria calcarea, Cir.cal; Circinaria contorta, Cir.con; Xanthocarpia crenulatella, 558
Xan.cre; Pyrenodesmia erodens, Pyr.ero; ) through the quadrats of the 30×150 cm plots (1-18; 559
black squares indicate the species occurrence). b Total frequency of the species through the 560
balustrade (% of plots which host the species; contribution to the species abundance from 561
plots in the S, dark grey, SW, grey, NW, light grey, N, white, parts of the balustrade). c 562
Average frequency per plot of each species (av. % of quadrats which host the species; 563
according to Tukey’s test, columns which do not share at least one letter are statistically 564
different, P<0.05). d Moran I’s coefficient (*, P<0.05). e Spread Index (according to Tukey’s 565
test, columns which do not share at least one letter are statistically different, P<0.05). 566
Fig. 3. Dispersion of reproductive structures by the surveyed species (abbreviations as in Fig. 567
2). a Ejected ascospores (per apothecium, as evaluated directly on the balustrade of the 568
Basilica of Superga): boxplots showing maxima values (upper whisker), 75th percentile (top 569
box), median (transversal line), 25th percentile (bottom box), minimum (lower whisker), 570
outliers (circles). b Ascospores dispersed at short distance and long distance (per apothecium, 571
as evaluated in the laboratory microcosm): maximum (black symbols) and minimum (white 572
symbols) values of ascospores collected within the passive traps at short distance from the 573
incubated ascocarps (squares) and ascospores taken off and collected by the Hirst-type 574
volumetric sampler, available for long distance dispersal (circles). Reproductive structures of 575
Pyrenodesmia erodens, lacking ascocarps, but showing soredia, were never collected, neither 576
in ejection nor in dispersal assays. 577
Fig. 4. Ascospores collected in ejection and dispersal assays. a-c Packages of spores collected 578
in ejection tests with Candelariella aurella (a), Verrucaria macrostoma (b) and Xanthocarpia 579
crenulatella (c). Bars = 20 μm. d-e Ascospores of Circinaria contorta within an ascocarp (*, 580
d) and collected with the passive traps at a short distance from the reproductive structures 581
incubated in the laboratory microcosm (e). f-g Ascospores of Xanthocarpia crenulatella 582
collected by the Hirst volumetric sampler in the laboratory microcosm (f) and within an 583
ascocarp (*, g). Scale bars: 20 µm (a-e, g), 10 µm (f). 584
Fig. 5. Factorial map of RDA carried out on population structure parameters (total frequency 585
through the plots, av. frequency per plot; Moran I’s coefficient; Spread Index), dispersal 586
(maxima values of ejected spores, and spores involved in short- and long distance dispersal) 587
and microenvironment-related (CorrSol, CorrTree) parameters. a Axes 1 and 2. b Axes 1 and 588
3. Species abbreviations as in Fig. 2. 589
Table 1. Ecological indicator values (Nimis 2016) of lichen species recorded on the balustrade of the Basilica of Superga, and Pearson correlation coefficients (ρ) of species frequency per plot with solar exposure (CorrSol) and distance from nearest trees (CorrTree). Indicator scale for Light (Nimis 2016): 1 - in very shaded situations, 2 - in shaded situations, 3 - in sites with plenty of diffuse light but scarce direct solar irradiation, 4 - in sun-exposed sites, but avoiding extreme solar irradiation, 5 - in sites with very high direct solar irradiation; indicator scale for Eutrophication (Nimis 2016): 1 - not resistent to eutrophication; 2 - resistent to a very weak eutrophication; 3 - resistent to a weak eutrophication; 4 - occurring in rather eutrophicated situations; 5 - occurring in highly eutrophicated situations. Significant correlations according to Bonferroni’s test (P<0.05) are reported in bold.
Species
Ecol. indicator values CorrSol CorrTree
Light Eutrophication ρ P ρ P Candelariella aurella 3-5 2-4 -0.273 1.000 -0.463 0.422 Myriolecis gr. dispersa 3-5 2-4 -0.637 0.160 -0.405 0.766 Myriolecis albescens 3-5 3-4 -0.560 0.566 -0.226 1.000 Verrucaria macrostoma 3-4 3-5 0.333 1.000 -0.208 1.000 Circinaria calcarea 4-5 2-3 -0.765 0.008 0.060 1.000 Circinaria contorta 3-4 3-5 -0.437 1.000 -0.223 1.000 Xanthocarpia crenulatella 4 4 0.440 1.000 -0.524 0.205 Pyrenodesmia erodens 4-5 2-3 0.493 1.000 0.111 1.000 Table 1
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